Polyethylene glycol (PEG) is a polymer having the structure H(O—CH2CH2—)nOH. It is generally synthesized by the ring opening polymerization of ethylene oxide. While the polymer usually has a linear structure at molecular weights ≦10 kD, the higher molecular weight PEGs may have some degree of branching.1 Polyethylene glycols of different molecular weights have previously been used in a number of applications, including to increase the solubility of drugs. During the last three decades, polyethylene glycol has been extensively investigated for delivery of various proteins via parenteral routes. Generally, PEGs have been most widely used for the delivery of both traditional drugs (small molecules) and proteins/enzymes in the treatment of cancer.
Several chemical procedures have been developed for the preparation of activated PEGs, which can then be used to react specifically with free amino groups on an enzyme's surface, under mild aqueous conditions. PEGs have been successfully activated by reaction with 1,1-carbonyl-di-imidazole, cyanuric chloride, tresyl chloride, 2,4,5-trichlorophenyl chloroformate or 4-nitrophenyl chloroformate, various N-hydroxy-succinimide derivatives as well as by the Moffatt-Swern reaction.2-10 In most cases, the activating agent acts as a linker between the PEG and the enzyme or protein.
One of the major disadvantages encountered with the processes currently available involves the reaction temperatures at which the activation reactions are carried out. The most commonly used solvents are acetonitrile (CH3CN) and dichloromethane (CH2Cl2), containing small volumes of a co-solvent, usually triethylamine (TEA). Usually, the activation reactions are carried out under refluxing conditions at a temperature of about 84° C. when acetonitrile is used, or at a temperature of about 40° C. when dichloromethane is chosen as the solvent.
Several crystallization steps are commonly required for the isolation and purification of the activated PEG product. Such steps can make currently available processes for the activation of PEGs inconvenient.
The activation of PEGs with 4-nitrophenyl chloroformate, to generate PEG-di-nitrophenyl carbonates, has been described by Fortier and Laliberté.10 The reactions were carried out in acetonitrile containing triethylamine (TEA) over a period of 5 hours at 60° C. The long reaction times and the reaction temperatures required to perform the activation reactions are major disadvantages of this process. Additionally, in order to keep the system as anhydrous as possible, the use of a cumbersome soxhlet is required. This imparts a severe limitation on the activation process, especially when transposed on a larger scale.
There is thus a need for a low-cost process for the activation of PEGs that is time efficient and that can be performed at room temperature. There is also a need for a process allowing for the rapid isolation and purification of the activated PEGs.
Moreover, there is a need for a process for PEG activation that is amenable to large-scale production.
The present invention seeks to meet these and other needs.